Establishment Of Glioma Bank And Expression And Significance Of EphB4,CD105 In Glioma

Objective: Along with research of molecular biology of glioma, the requirement of related research institute to each kind of tumor grows day by day, especially after the beginning of postgenome plan, this requirement even more urgency.The formerly researcher is only according to the need of some research topic to collect tumor specimen, many valuable tumor specimens and related data are lost, the loss of tumor research is large.The objective and significance of establishing glioma bank is to provide the specimen origin and patient data for the research of preclinical medicine,clinical medicine and molecular biology.Glioma is the most frequent neuroepithelium tumor, even though we take operation,radiotherapy and chemotherapy, the therapeutic effect is still bad and the life span is still short. Ephs acceptors are the factor of directing neuron deadexis, later we discover that it is the key factor to control the forming of blood vessel and embryo, recently we find that it participates the forming of tumor. Recently year, along with the research of TGF-β,we find it can inhibit the growth of endothelial cell out of body, but in vivo it can integrate inflammatory cell and induce it to release angiogenesis factor and stimulate endothelial cell division growth, meanwhile it can effect the cell differ- entiation,intercellular substance metabolism and immune function. The function of TGF-βis implemented for integrating specific acceptors, CD105 is one of them. In light of the close relationship between tumor and new vessels, EphB4 and CD105 have double function, so this experiment is to research the expression of EphB4 and CD105 in glioma, and attempt to establish glioma bank.Methods: The case group involved fifty glioma patients'specimen who were after operation in neurosurgery in the second hospital of Hebei Medical University from 2006.12- 2007.6 (34 of them are male,16 of them are female), ages are between 17 and 75, average age is 46.82±14.69, 5 normal brain specimens are used to be the contrast. According to the WHO separate criteria, 27 of them are gradeⅡ,13 of them are gradeⅢ,10 of them are gradeⅣ. We use immunohistochemistry and semiquantitative RT-PCR technology to detect the expression of EphB4 and CD105 in all the specimens and count the micro- vessel density(MVD).Judgment of IHC result: first, two persons count 1000 tumor cells under the high power lens, according to the percentage of positive tumor cell, we divide 4 grade: gradeⅠ,0 score,<10%; gradeⅡ,1 score, 10%～25%; gradeⅢ,2 scores, 25%～50%; gradeⅣ,3 scores,>50%. According to the chromatosis is shade or not, we score the magnitude of staining: 1 score, poor; 2 scores, middling; 3 scores, strong. Plus above of two scores are the tumor specimen's IHC positive score: negative, 0～1 score, we sign (-); weakly positive, 2～4 scores, we sign(+); positive,≥5 scores, we sign(++).Method of counting MVD: If we find a endothelial cell in the tumor tissue, we sign one positive blood vessel marker, we observe one slice of every specimens, avoid the tumor necrosis region or bleeding region, we select the most abundant area of micrangium , which is'hot spot', we count the average of blood vessels under five high power fields(400×). We use (X|-)±S to demonstrate the MVD of glioma of different series.Statistical method: we choose Kruskal-Wallis H exami- nation,Nemenyi examination of two samples,one-way ANOVA and Spearman rank correlation analysis. We use SPSS14.0 statistics software to processing data. When P<0.05, the data has statistics significance.Results: 1. IHC result: In 5 normal brain organizations, EphB4 and CD105 protein expression are the negative or weak expression. In 50 gliomas, EphB4 locates in the tumor cell membrane,CD105 locates in the vascular endothelial cell me- mbrane, EphB4 and CD105 positive and the strong positive expression respectively is 37 examples and 25 examples, the expression level increases along with the tumor malignant degree elevates, the expressions of EphB4 between normal brain and gradeⅡ,gradeⅢandⅣhave not remarkable differences, differences between other two groups are all have the signi- ficance(P<0.05), the expression intensity and the glioma path- ology graduation present positive correlation.(r_EphB4= 0.608, r_CD105=0.856, p=0.000)2. RT-PCR result: 1.2% agarose gelatin result show that we can obtain EphB4 and the CD105 PCR product signal res- pectively about molecular weight 139bp and 396, normal brain organization is low expression, the different rank glioma's expression level increases along with the tumor malignant degree elevates, we use non-RNase water substitutes the AMV retroviridase to be the negative comparison. The differences between the two groups are all have the significance(P<0.05), the expression intensity and the glioma pathology graduation present positive correlation.(r_EphB4=0.899,r_CD105=0.904,p=0.000). we can obtainβ-actin PCR product signal about molecular weight 500bp. The product signal in normal brain organization and in each rank glioma shows the high expression, the expression level is approximately equal.Conclusion: 1. EphB4 shows the high expression in the glioma, and increases along with the tumor degree elevates, the expression intensity and the glioma pathology graduation present positive correlation.2. CD105 also shows the high expression in the glioma, and increases along with the tumor degree elevates, the expression intensity and the glioma pathology graduation present positive correlation.3. EphB4 and CD105 have close correlation with the differ- rentiation degree of glioma and the forming of blood vessel, the excessive expression play the vital role in the development of tumor and the forming of blood vessel, they reflect growth infiltration ability of tumor cell from two different sides of proliferation activity and blood vessel production. EphB4 and CD105 are good indexes for reference, we can use them to judge the malignant degree and pathology characteristic of tum- ors.